The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of this proposal is to recapitulate the T. gondii sexual cycle in tissue culture. Creation of ex vivo sexual development conditions will allow the contribution of specific genes to biological processes and redundant pathways to be rapidly analyzed. It will also allow for a molecular analysis of the complete lifecycle of a protistan parasite, as T. gondii asexual development can already be performed in tissue culture. Another benefit of tissue culture sexual development of T. gondii will be the stable production of a T. gondii vaccine vector against itself and other pathogens, such as Plasmodium. Recombinant T. gondii expressing circumsporozoite protein from Plasmodium yoelii or P. knowlesi provides protective immunity against malaria infection in mice and rhesus monkeys, respectively. A T. gondii vaccine vector produced as an oocyst will be ideal because it will be stable in virtually any environmental condition and it will be an oral inoculation. My laboratory is also identifying the virulence genes critical for T. gondii to establish a chronic infection. Synergizing the work from these proposals will allow us to create an attenuated vaccine strain that can be produced as a stable oocyst from cell culture. PUBLIC HEALTH RELEVANCE: The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of this proposal is to recapitulate the T. gondii sexual cycle in tissue culture. Another benefit of tissue culture sexual development of T. gondii will be the stable production of a T. gondii vaccine vector against itself and other pathogens, such as Plasmodium.